Real time imaging of blood vessels that reveals the structure and hemodynamics of the vascular system has a variety of biomedical applications (see, for example, Makale, M., Methods in Enzymology 2008, 444, 175-199; Ghaffari, S., Development 2015, 142, 4158-4167; Sahn, D. and Vick, G., Heart 2001, 86 (Supp) II), ii41-ii53). Several imaging technologies are currently available for these applications (for a review, see Upputuri, P., BioMed Research International, 2015, Article ID 783983, and references therein). However, they all suffer from one or more of a number of disadvantages such as equipment cost, complicated procedure, harmful exposure of test subjects to high energy, long scanning and post-process time, or insufficient resolution. These limitations are particularly prohibitory against large-scale use on animals in preclinical studies. A quick, easy-to-operate, low cost, and high resolution vasculature imaging technology, applicable to live animals, i.e. being vital, in particular, is not only a desirable improvement for clinical applications, but will prove highly useful for studying human diseases in animal models.
The value of such technologies is well demonstrated in cancer. Angiogenesis is a hallmark of cancer in which tumor cells in patients recruit new blood vessels to supply nutrients for tumor growth and metastasis (see Carmeliet, P. and Jain, R., Nature 2000, 407, 249-257). Therefore, detection, monitoring, and inhibition of angiogenesis surrounding cancer cells play a critical role in our fight against cancer. In the area of anti-cancer therapy development, there is continuous interest in tools and methods that characterize the occurrence of angiogenesis and enable the discovery of agents that inhibit or promote angiogenesis and thus tumor progression.
In vitro assays are available to examine pro- and anti-angiogenic substances (see, for examples, Ngo, T. et. al., International Journal of Tissue Regeneration 2014, 5(2), 37-45; Ucuzian, A and Greisler, H., World J. Surg. 2007, 31, 654-663). These assays are designed to mimic the in vivo environment. However, their adequacy is open to debate and the results from these assays always require confirmation in vivo.
In vivo assays for angiogenesis that use a specific part or system of live animals also have a long history of development. These models are mostly performed on rodent or larger animals, such as dogs, and can be prohibitively expensive. They are all technically cumbersome due to the need for surgery and the operations are often time consuming (see Norrby, K., J. Cell. Mol. Med. 2006, 10(3), 588-612).
Whole animal models have been developed to study angiogenesis using zebrafish (Danio rerio), the Xenopus Laevis tadpole, and more recently the invertebrate Hirudo medicinalis (see, for example, Ny, A. et. al., Experimental Cell Research 2006, 312, 684-693). For reasons highlighted in the following, the zebrafish models have stood out in particular, in terms of practicality, for the research on angiogenesis and its modulators.
First of all, approximately 70% of all human protein-coding genes have functional homologs in zebrafish (see Howe, K. et. al., Nature 2013, 496, 498-503). Each mating pair of zebrafish produces hundreds of offspring per week, making embryos readily available for large scale phenotypic screening. Zebrafish embryos and early larvae are virtually transparent which makes visualization of its tissues and organs feasible. These and other features have placed zebrafish high on the list of model animals available for the investigation of human diseases and for the discovery of potential therapeutic drugs in general (see, for example, Lieschke, G. and Currie, P., Nature Reviews Genetics 2007, 8, 353-367; Santoriello, C. and Zon, L., J. Clin. Invest. 2012, 122(7), 2337-2343).
The vascular anatomy of developing zebrafish embryo has been described in detail. In addition to a high structural homology to other vertebrates, the molecular mechanisms underlying vessel formation in zebrafish are highly similar to those in humans and other higher vertebrates (see Isogai, S. et. al., Developmental Biology 2001, 230, 278-301). Early analysis of vascular pattern in zebrafish was performed by microinjection of dyes or small fluorescent microspheres into the blood vessels. The injection is difficult to perform due to the size of embryos, and mature vascular lumenization and connection to the circulation are required for the method to work. Imaging of zebrafish blood vessels can also be done by staining for endogenous alkaline phosphatase (AP) activity in vascular endothelium, but only within a fix development window when the AP signal is high relative to background staining. All these methods are performed after specimen fixation; they are not vital and cannot be used to show the dynamics of vessel development or blood flow (see Kamei, M. et. al., Methods in Cell Biology 2010, 100, 27-54).
So far, the most convenient vital imaging of zebrafish vascular system relies on expression of vascular specific transgenic fluorophores. Numerous transgenic zebrafish lines have been engineered and generated that express a vascular specific gene with green fluorescence protein as a reporter in related tissues for the visualization and study of zebrafish vascular development (see Chavez, M. et. al., Front. Physiol. 2016, 7, article 56). These mutant fishes have been widely used to study the effect of treatments on the development of blood vessels. The drawback is that the fluorescence is inherited and permanent once developed. And the results cannot be related to wild type without the inherent risks of using inference.